Methods and systems for fast pcr heating

ABSTRACT

A microplate for polymerase chain reaction (PCR) comprises a substrate having a metallic material for heating PCR samples, and a barrier layer disposed adjacent to the substrate. In some cases, the barrier layer is formed of a first polymeric material. The microplate includes one or more wells for containing PCR samples. The one or more wells are formed of a second polymeric material sealed to the barrier layer. In some cases, the substrate can provide a PCR ramp rate of at least about 5° C./second.

CROSS-REFERENCE

This application in a continuation application of patent applicationSer. No. 13/329,183 filed on Dec. 16, 2011, which claims the benefit ofU.S. Provisional Patent Application No. 61/424,551, filed Dec. 17, 2010and U.S. Provisional Patent Application No. 61/545,063, filed Oct. 7,2011, each of which is entirely incorporated by reference.

BACKGROUND OF THE INVENTION

In many fields specimen carriers in the form of support sheets, whichmay have a multiplicity of wells or impressed sample sites, are used forvarious processes where small samples are heated or thermally cycled. Aparticular example is the Polymerase Chain Reaction method (oftenreferred to as PCR) for replicating DNA samples. Such samples requirerapid and accurate thermal cycling, and are typically placed in amulti-well block and cycled between several selected temperatures in apre-set repeated cycle. It is important that the temperature of thewhole of the sheet or more particularly the temperature in each well beas uniform as possible.

The samples may be liquid solutions, typically between 1 micro-1 and 200micro-1 in volume, contained within individual sample tubes or arrays ofsample tubes that may be part of a monolithic plate. The temperaturedifferentials that may be measured within a liquid sample increase withincreasing rate of change of temperature and may limit the maximum rateof change of temperature that may be practically employed.

Previous methods of heating such specimen carriers have involved the useof attached heating devices or the use of indirect methods whereseparately heated fluids are directed into or around the carrier.

The previous methods of heating suffer from the disadvantage that heatis generated in a heater that is separate from the specimen carrier thatis required to be heated. Such heating systems and methods suffer fromheat losses accompanying the transfer of heat from the heater to acarrier sheet of the specimen carrier. In addition, the separation ofthe heater from the specimen carrier introduces a time delay or “lag” inthe temperature control loop. Thus, the application of power to theheating elements does not produce an instantaneous or near instantaneousincrease in the temperature of the block. The presence of a thermal gapor barrier between the heater and the block requires the heater to behotter than the block if heat energy is to be transferred from theheater to the block. Therefore, there is a further difficulty thatcessation of power application to the heater does not instantaneouslystop the block from increasing in temperature.

The lag in the temperature control loop will increase as the rate oftemperature change of the block is increased. This may lead toinaccuracies in temperature control and limit the practical rates ofchange of temperature that may be used. Inaccuracies in terms of thermaluniformity and further lag may be produced when attached heatingelements are used, as the elements are attached at particular locationson the block and the heat produced by the elements must be conductedfrom those particular locations to the bulk of the block. For heattransfer to occur from one part of the block to another, the first partof the block must be hotter than the other. Another problem withattaching a thermal element, particularly current Peltier effectdevices, is that the interface between the block and the thermal devicewill be subject to mechanical stresses due to differences in the thermalexpansion coefficients of the materials involved. Thermal cycling willlead to cyclic stresses that will tend to compromise the reliability ofthe thermal element and the integrity of the thermal interface.

SUMMARY OF THE INVENTION

An aspect of the invention provides a microplate for polymerase chainreaction (“PCR”), comprising a substrate comprising a metallic materialfor heating PCR samples and a barrier layer disposed adjacent to thesubstrate, the barrier layer formed of a first polymeric material. Themicroplate includes one or more wells for holding PCR samples, the oneor more wells formed of a second polymeric material sealed to thebarrier layer. The substrate provides a PCR ramp rate of at least 5°C./second (“s”). In an embodiment, the PCR ramp rate (or heating rate)is at least about 10° C./second. In another embodiment, the microplateis configured to heat samples upon the flow of electric current throughthe substrate. In another embodiment, the substrate is configured to beseparated from PCR samples by 10 micrometers or less. In anotherembodiment, the second polymeric material is heat-sealed to the barrierlayer. In another embodiment, the first polymeric material is chemicallycompatible with the second polymeric material. In another embodiment,the metallic material comprises an aluminum alloy. In anotherembodiment, the substrate is for generating heat upon the flow ofelectrical current through the substrate. In another embodiment, thesubstrate is for increasing the temperature of a sample in the one ormore wells at a rate between about 5° C./s and 15° C./s. In anotherembodiment, the metallic material has a resistivity between about 2×10⁻⁸ohm-m and 8×10⁻⁸ ohm-m. In another embodiment, the one or more wellscomprise at least 24 wells. In another embodiment, the one or more wellscomprise at least 96 wells.

Another aspect of the invention provides a microplate for PCR,comprising a substrate comprising a metallic material for heating PCRsamples, and a coating layer disposed adjacent to the substrate, thecoating layer formed of a first polymeric material. The microplateincludes one or more wells formed of a second polymeric material sealedto the coating layer for containing PCR samples. The metal substrateprovides well-to-well thermal uniformity of +/−2° C., +/−1° C., +/−0.5°C., +/−0.2° C., +/−0.1° C., +/−0.05° C. or better without an externalheating element or a Peltier heating block.

Another aspect of the invention provides a microplate for PCR,comprising a substrate comprising a metallic material for heating PCRsamples, and a coating layer disposed adjacent to the substrate, thecoating layer formed of a first polymeric material. The microplateincludes one or more wells for containing PCR samples, the one or morewells formed of a second polymeric material sealed to the coating layer.The substrate provides a heating efficiency sufficient to allow for atleast about 1 PCR cycle per minute, including fluorescence measurementfor every cycle. In an embodiment, the substrate provides a heatingefficiency sufficient to allow for at least about 0.1, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,1000 or more PCR cycles per minute. In another embodiment, the substratecomprises an aluminum alloy. In another embodiment, the substratecomprises aluminum. In another embodiment, the microplate has athickness of less than about 1 millimeter (“mm”). In another embodiment,the microplate has a thickness of less than about 0.5 mm. In anotherembodiment, the coating layer has a thickness of less than about 10micrometers (“microns”). In another embodiment, the microplate furthercomprises a layer of an infrared radiation-normalizing layer at a sideof the substrate opposite the coating layer. In another embodiment, theradiation-normalizing layer has a thickness of less than 5 microns.

Another aspect of the invention provides a disposable sample holder foruse with PCR, comprising an aluminum substrate coated with a firstpolymeric material and a plurality of wells heat-sealed to the firstpolymeric material, the plurality of wells formed of a second polymericmaterial compatible with the first polymeric material.

Another aspect of the invention provides a disposable sample holder foruse with PCR having an aluminum-containing substrate for providing heatto a plurality of wells of the disposable sample holder, the disposablesample holder having a weight less than or equal to about 30 g. In anembodiment, the disposable sample holder has a weight less than or equalto about 20 g. In another embodiment, the disposable sample holder has aweight less than or equal to about 15 g. In another embodiment, thedisposable sample holder has a weight less than or equal to about 10 g.In another embodiment, the disposable sample holder has a weight lessthan or equal to about 5 g.

Another aspect of the invention provides a low-cost sample holder foruse with PCR, comprising a substrate formed of a metallic materialhaving a density between about 2.7 g/cm³ and 3.0 g/cm³. The substrate isconfigured to provide heat to one or more wells of the low-cost sampleholder at a heating rate between about 5° C./s and 15° C./s. In anembodiment, the substrate comprises aluminum. In another embodiment, thelow-cost sample holder further comprises a barrier layer formed of afirst polymeric material over the substrate. In another embodiment, theone or more wells are formed of a second polymeric material joined tothe first polymeric material.

Another aspect of the invention provides a microplate for PCR,comprising a Peltier heating device and a substrate adjacent to thePeltier heating device, the substrate comprising a metallic material forheating PCR samples. The microplate further comprises a barrier layeradjacent to the substrate, the barrier layer formed of a first polymericmaterial. One or more wells are disposed adjacent to the substrate, theone or more wells for containing PCR samples. The one or more wells areformed of a second polymeric material sealed to the barrier layer. In anembodiment, the first polymeric material is different from the secondpolymeric material. In another embodiment, the first polymeric materialis the same as the second polymeric material. In another embodiment, thesubstrate provides a PCR ramp rate of at least 5° C./second. In anotherembodiment, the substrate provides a PCR ramp rate of at least 10°C./second.

Another aspect of the invention provides a microplate for PCR,comprising a substrate comprising a metallic material for heating PCRsamples and a barrier layer adjacent to the substrate, the barrier layerformed of a first polymeric material. The microplate further comprisesone or more wells adjacent to the substrate, the one or more wells forcontaining PCR samples. The one or more wells are formed of a secondpolymeric material sealed to the barrier layer. The microplate isconfigured to come in electrical contact with one or more electricalcontact (e.g., bus bars) at one or more corrugated surfaces of thesubstrate. In an embodiment, the first polymeric material is differentfrom the second polymeric material. In another embodiment, the firstpolymeric material is the same as the second polymeric material. Inanother embodiment, the one or more corrugated surfaces are disposed atfinger-like projections (also “fingers” herein) of the substrate. Inanother embodiment, the one or more corrugated surfaces are formed fromthe fingers. In another embodiment, the substrate provides a PCR ramprate of at least 5° C./second. In another embodiment, the substrateprovides a PCR ramp rate of at least 10° C./second.

Another aspect of the invention provides a microplate for PCR,comprising a heating device and a substrate adjacent to the heatingdevice, the substrate comprising a metallic material for heating PCRsamples upon the flow of current through the substrate. The microplatefurther comprises a barrier layer adjacent to the substrate, the barrierlayer formed of a first polymeric material. One or more wells aredisposed adjacent to the substrate, the one or more wells for containingPCR samples. The one or more wells are formed of a second polymericmaterial sealed (e.g., heat sealed, clamped) to the barrier layer. In anembodiment, the heating device is a Peltier heating device or a heatingclamp. In another embodiment, the first polymeric material is differentfrom the second polymeric material. In another embodiment, the firstpolymeric material is the same as the second polymeric material.

Another aspect of the invention provides a method for conducting PCR,wherein data from the PCR and instructions for processing the data arelocated on a removable device. In an embodiment, both control andanalysis instructions are provided in the removable device to allow auser to develop an experiment and analyze the results independently froma thermal cycler used for conducting PCR. In another embodiment, theremovable device is a universal serial bus device. In anotherembodiment, the removable device is a removable memory disk. In anotherembodiment, the removable device is a compact flash, a serial advancedtechnology attachment interface, or a personal computer memory cardinternational association interface. In another embodiment, theinstructions on the removable device enable an identification of thetype of hardware interfacing with the removable device and providepredetermined commands and/or instructions for performing PCR on thehardware.

Another aspect of the invention provides a system for performing PCR,comprising a plurality of bus bars for electrically mating with amicroplate or sample holder, and a microplate or sample holder asdescribed above or elsewhere herein, alone or in combination. Themicroplate or sample holder is in electrical communication with, andremovable from, the plurality of bus bars. The system further comprisesa current application device for applying current to the microplate orsample holder. In an embodiment, the microplate or sample holder is inohmic contact with the plurality of bus bars. In another embodiment, themicroplate or sample holder comprises finger-like projections inelectrical communication (or electrical contact) with the bus bars. Inanother embodiment, the finger-like projections have surfaces comprisingcrinkles. In another embodiment, the system further comprises atemperature sensor such as an infrared sensor, for measuring thetemperature of the microplate or sample holder. In another embodiment,the system further comprises a plurality of temperature sensors formeasuring the temperature of the microplate or sample holder in aplurality of thermal zones. In another embodiment, the system comprisesat least nine sensors for measuring the temperature of the microplate orsample holder in nine thermal zones. In another embodiment, theplurality of temperature sensors provide continuous temperaturemeasurements. In another embodiment, temperature variation across themicroplate or sample holder is less than about 0.5° C.

Another aspect of the invention provides a method for conducting PCR,comprising providing a microplate or sample holder as described above orelsewhere herein, alone or in combination, and conducting PCR on thesample. During PCR, the sample is heated at a ramp rate of at leastabout 5° C./second. In an embodiment, the method further comprisesproviding a sample to the microplate or sample holder before conductingPCR. In an embodiment, during PCR the sample is heated at a ramp rate ofat least about 0.1° C./second. In another embodiment, during PCR thesample is heated at a ramp rate of at least about 0.5° C./second. Inanother embodiment, during PCR the sample is heated at a ramp rate of atleast about 1° C./second. In another embodiment, during PCR the sampleis heated at a ramp rate of at least about 5° C./second. In anotherembodiment, during PCR the sample is heated at a ramp rate of at leastabout 10° C./second. In another embodiment, during PCR the sample isheated at a ramp rate of at least about 15° C./second.

Another aspect of the invention provides a method for performing PCR,comprising providing a microplate or sample holder as described above orelsewhere herein, alone or in combination, and conducting PCR on thesample. During PCR, the microplate or sample holder has well-to-wellthermal uniformity of at least about +/−2° C., +/−1° C., +/−0.5° C.,+/−0.2° C., +/−0.1° C., +/−0.05° C. without an external heating elementor a Peltier heating block. In an embodiment, the method furthercomprises providing a sample to the microplate or sample holder beforeconducting PCR.

Another aspect of the invention provides a method for conducting PCR,comprising providing a microplate or sample holder as described above orelsewhere herein, alone or in combination, and conducting PCR on thesample at a rate of at least about 0.1 PCR cycles per minute. In anembodiment, the method further comprises providing a sample to themicroplate or sample holder before conducting PCR. In anotherembodiment, PCR is conducted on the sample at a rate of at least about 1PCR cycle per minute. In another embodiment, PCR is conducted on thesample at a rate of at least about 2 PCR cycles per minute. In anotherembodiment, PCR is conducted on the sample at a rate of at least about 3PCR cycles per minute. In another embodiment, PCR is conducted on thesample at a rate of at least about 6 PCR cycles per minute. In anotherembodiment, the method further comprises performing fluorescencemeasurement in an individual PCR cycle. In another embodiment, duringPCR, the microplate or sample holder has well-to-well thermal uniformityof at least about +/−2° C., +/−1° C., +/−0.5° C., +/−0.2° C., +/−0.1°C., +/−0.05° C. without an external heating element or a Peltier heatingblock. In another embodiment, during PCR the sample is heated at a ramprate of at least about 0.1° C./second. In another embodiment, during PCRthe sample is heated at a ramp rate of at least about 0.5° C./second. Inanother embodiment, during PCR the sample is heated at a ramp rate of atleast about 1° C./second. In another embodiment, during PCR the sampleis heated at a ramp rate of at least about 5° C./second. In anotherembodiment, during PCR the sample is heated at a ramp rate of at leastabout 10° C./second. In another embodiment, during PCR the sample isheated at a ramp rate of at least about 15° C./second.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic side-view of a microplate for polymerase chainreaction (PCR), in accordance with an embodiment of the invention;

FIG. 2 schematically illustrates a transformer drive pattern forproviding heat to a consumable, in accordance with an embodiment of theinvention;

FIG. 3 schematically illustrates a transformer drive pattern forproviding heat to a consumable, in accordance with an embodiment of theinvention;

FIG. 4 schematically illustrates a transformer drive pattern forproviding heat to a consumable, in accordance with an embodiment of theinvention;

FIG. 5 schematically illustrates a transformer drive pattern forproviding heat to a consumable, in accordance with embodiments of theinvention;

FIG. 6 shows a sensor block, in accordance with an embodiment of theinvention;

FIG. 7 shows a Peltier heating device, in accordance with an embodimentof the invention;

FIG. 8 shows a microplate and a Peltier heating device adjacent to themicroplate, in accordance with an embodiment of the invention;

FIG. 9 shows a system for performing PCR, in accordance with anembodiment of the invention;

FIG. 10 shows a microplate having 54 wells, in accordance with anembodiment of the invention; and

FIGS. 11-17 illustrate exemplary screenshots of a graphical userinterface, in accordance with an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

While preferable embodiments of the invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

In embodiments, microplate assemblies (also “microplates” herein) areprovided for polymerase chain reaction (PCR). Microplates of embodimentsof the invention may provide various advantages over current PCRsystems, as rapid and accurate thermal control during PCR. In someembodiments, microplates are provided that can perform at least about0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 1000 PCR cycles per minute, in some cases withfluorescence measurements every cycle. In another embodiment,microplates are provided having an average heating ramp rate of at leastabout 0.01° C./second, or 0.1° C./second, or 1° C./second, or 2°C./second, or 3° C./second, or 4° C./second, or 5° C./second, or 6°C./second, or 7° C./second, or 8° C./second, or 9° C./second, or 10°C./second, or 11° C./second, or 12° C./second, or 13° C./second, or 14°C./second, or 15° C./second, or 16° C./second, or 17° C./second, or 18°C./second, or 19° C./second, or 20° C./second, or 25° C./second, or 30°C./second, or 35° C./second, or 40° C./second, or 45° C./second, or 50°C./second, 100° C./second, or more. In another embodiment, microplatesare provided having active control over thermal uniformity, producingthermal control to within +/−2° C., +/−1° C., +/−0.5° C., +/−0.2° C.,+/−0.1° C., +/−0.05° C. or better.

In some embodiments, microplates may be consumable. In anotherembodiment, microplates may be recyclable. In another embodiment,microplates may be reusable. In another embodiment, microplates may bebiodegradable. In another embodiment, a microplates may benon-consumable.

Microplates for Polymerase Chain Reaction (PCR)

An aspect of the invention provides a microplate for polymerase chainreaction (PCR). In embodiments, the microplate comprises a substrateincluding a metallic material for heating PCR samples and a barrierlayer disposed over the substrate, the barrier layer formed of a firstpolymeric material. The microplate further includes one or more wellsfor containing PCR samples, the one or more wells formed of a secondpolymeric material sealed to the barrier layer. In some cases, the firstpolymeric material is different from the second polymeric material. Inan example, the first polymeric material has a different glasstransition temperature than the second polymeric material. In othercases, the first polymeric material is the same as the second polymericmaterial. In an example, the first polymeric material has the same orsubstantially the same glass transition temperature as the secondpolymeric material.

In some embodiments, the substrate provides a PCR ramp rate (or heatingrate) of at least about 0.01° C./second, or 0.1° C./second, or 1°C./second, or 2° C./second, or 3° C./second, or 4° C./second, or 5°C./second, or 6° C./second, or 7° C./second, or 8° C./second, or 9°C./second, or 10° C./second, or 11° C./second, or 12° C./second, or 13°C./second, or 14° C./second, or 15° C./second, or 16° C./second, or 17°C./second, or 18° C./second, or 19° C./second, or 20° C./second, or 25°C./second, or 30° C./second, or 35° C./second, or 40° C./second, or 45°C./second, or 50° C./second, or 100° C./second, or more.

In some embodiments, heating of PCR samples may be achieved by passingelectric current through the substrate. In another embodiment, heatingof PCR samples may be achieved by passing direct current (DC) throughthe substrate. In another embodiment, heating of PCR samples may beachieved by passing alternating current (AC) through the substrate.

In some embodiments, the substrate is separated from a PCR sample by 1micrometer (“micron”) or less, or 2 microns or less, or 3 microns orless, or 4 microns or less, or 5 microns or less, or 6 microns or less,or 7 microns or less, or 8 microns or less, or 9 microns or less, or 10microns or less, or 11 microns or less, or 12 microns or less, or 13microns or less, or 14 microns or less, or 15 microns or less, or 16microns or less, or 17 microns or less, or 18 microns or less, or 19microns or less, or 20 microns or less. In other embodiments, thesubstrate is separated from a PCR sample by at least about 0.1 microns,or 1 micron, or 2 microns, or 3 microns, or 4 microns, or 5 microns, or10 microns, or 15 microns, or 20 microns, or 30 microns, or 40 microns,or 50 microns, or 100 microns, or 500 microns, or 1000 microns, or 5000microns, or 10,000 microns, or more.

In some embodiments, the second polymeric material is heat-sealed to thebarrier layer. In another embodiment, the first polymeric material ischemically compatible with the second polymeric material. In anotherembodiment, the metallic material comprises aluminum or an aluminumalloy.

In some embodiments, the substrate is for generating heat upon the flowof electrical current through the substrate. In another embodiment, thesubstrate is for generating heat upon the flow of direct current (DC)through the substrate. In another embodiment, the substrate is forgenerating heat upon the flow of alternating current (AC) through thesubstrate.

In some embodiments, the substrate is for increasing the temperature ofa sample in the one or more wells at a rate between about 0.01°C./second and 100° C./second, or between about 0.1° C./second and 50°C./second, or between about 1° C./second and 35° C./second, or betweenabout 3° C./second and 25° C./second, or between about 5° C./second and15° C./second.

In some embodiments, the substrate includes a metallic material forheating PCR samples. The metallic material may have a resistivitybetween about 5×10⁻⁹ ohm-m and 1×10⁻⁶ ohm-m, or between about 1×10⁻⁸ohm-m and 1×10⁻⁷ ohm-m, or between about 2×10⁻⁸ ohm-m and 8×10⁻⁸ ohm-m.

In some embodiments, the microplate can include one or more wells. Insome cases, the microplate can include 1 well, or 2 wells, or 3 wells,or 4 wells, or 5 wells, or 6 wells, or 7 wells, or 8 wells, or 9 wells,or 10 wells, or 11 wells, or 12 wells, or 13 wells, or 14 wells, or 15wells, or 16 wells, or 17 wells, or 18 wells, or 19 wells, or 20 wells,or 21 wells, or 22 wells, or 23 wells, or 24 wells, or 25 wells, or 26wells, or 27 wells, or 28 wells, or 29 wells, or 30 wells, or 31 wells,or 32 wells, or 33 wells, or 34 wells, or 35 wells, or 36 wells, or 37wells, or 38 wells, or 39 wells, or 40 wells, or 41 wells, or 42 wells,or 43 wells, or 44 wells, or 45 wells, or 46 wells, or 47 wells, or 48wells, or 49 wells, or 50 wells, or 51 wells, or 52 wells, or 53 wells,or 54 wells, or 55 wells, or 56 wells, or 57 wells, or 58 wells, or 59wells, or 60 wells, or 61 wells, or 62 wells, or 63 wells, or 64 wells,or 65 wells, or 66 wells, or 67 wells, or 68 wells, or 69 wells, or 70wells, or 71 wells, or 72 wells, or 73 wells, or 74 wells, or 75 wells,or 76 wells, or 77 wells, or 78 wells, or 79 wells, or 80 wells, or 81wells, or 82 wells, or 83 wells, or 84 wells, or 85 wells, or 86 wells,or 87 wells, or 88 wells, or 89 wells, or 90 wells, or 91 wells, or 92wells, or 93 wells, or 94 wells, or 95 wells, or 96 wells, or 97 wells,or 98 wells, or 99 wells, or 100 wells, or 101 wells, or 102 wells, or103 wells, or 104 wells, or 105 wells, or 106 wells, or 107 wells, or108 wells, or 109 wells, or 110 wells, or 111 wells, or 112 wells, or113 wells, or 114 wells, or 115 wells, or 116 wells, or 117 wells, or118 wells, or 119 wells, or 120 wells, or 121 wells, or 122 wells, or123 wells, or 124 wells, or 125 wells, or 126 wells, or 127 wells, or128 wells, or 129 wells, or 130 wells, or more. In some embodiments, themicroplate can include 1 or more, or 5 or more, or 10 or more, or 15 ormore, or 20 or more, or 25 or more, or 30 or more, or 35 or more, or 40or more, or 45 or more, or 50 or more, or 60 or more, or 70 or more or80 or more, or 90 or more, or 100 or more, or 110 or more, or 120 ormore, or 130 or more, or 140 or more, or 150 or more, or 200 or more, or300 or more, or 400 or more, or 500 or more, or 1000 or more wells.

In an embodiment, the microplate may include 24 wells. In anotherembodiment, the microplate may include 48 wells. In another embodiment,the microplate can include 54 wells. In another embodiment, themicroplate may include 72 wells. In another embodiment, the microplatemay include 96 wells. The microplate can be disposable and/orrecyclable.

In some embodiments, the microplate may include 24 wells, each wellhaving a volume between 5 micro litre (μl) and 40 μl fill, or 96 wells,each well having a volume between about 0.5 μl and 5 μl.

In other embodiments, a microplate for polymerase chain reaction (PCR)comprises a substrate comprising a metallic material for heating PCRsamples, a coating layer (also “barrier layer” herein) disposed over thesubstrate, the coating layer formed of a first polymeric material; andone or more wells formed of a second polymeric material sealed to thecoating layer for containing PCR samples. In some embodiments, the metalsubstrate provides well-to-well thermal uniformity of +/−2° C. orbetter, or +/−1° C. or better, or +/−0.5° C. or better, or +/−0.2° C. orbetter, or +/−0.1° C. or better, or +/−0.05° C. or better, without theneed for an external heating element or a Peltier heating block.

In other embodiments, a microplate for polymerase chain reaction (PCR)comprises a substrate comprising a metallic material for heating PCRsamples; a coating layer disposed over the substrate, the coating layerformed of a first polymeric material; and one or more wells forcontaining PCR samples, the one or more wells formed of a secondpolymeric material sealed to the coating layer. In some embodiments, themetal substrate provides a heating efficiency sufficient to allow for atleast 0.1 PCR cycles per minute, or at least 1 PCR cycle per minute, orat least 2 PCR cycles per minute, or at least 3 PCR cycles per minute,or at least 4 PCR cycles per minute, or at least 5 PCR cycles perminute, or at least 6 PCR cycles per minute, or at least 7 PCR cyclesper minute, or at least 8 PCR cycles per minute, or at least 9 PCRcycles per minute, or at least 10 PCR cycles per minute, or at least 20PCR cycles per minute, or at least 30 PCR cycles per minute, or at least40 PCR cycles per minute, or at least 50 PCR cycles per minute, or atleast 60 PCR cycles per minute, or at least 70 PCR cycles per minute, orat least 80 PCR cycles per minute, or at least 90 PCR cycles per minute,or at least 100 PCR cycles per minute, or at least 200 PCR cycles perminute, or at least 300 PCR cycles per minute, or at least 400 PCRcycles per minute, or at least 500 PCR cycles per minute, or at least1000 PCR cycles per minute, in some cases including fluorescencemeasurement for every cycle.

In some embodiments, the microplate further includes a layer of aninfrared radiation (IR)-normalizing material at a side of the substrateopposite the contact layer. The IR normalizing layer may aid inincreasing IR emissivity, thereby providing for more efficient thermalregulation of the microplate and the one or more wells during PCR. Inanother embodiment, the microplate may comprise a layer of anIR-normalizing material at a side of the substrate opposite the coatinglayer. In some embodiments, the IR-normalizing layer may have athickness less than about 10 micrometers (“microns”), or less than about5 microns, or less than about 1 micron, or less than about 0.5 microns,or less than about 0.1 microns.

In some embodiments, the microplate may have a thickness less than about0.1 mm, or less than about 0.2 mm, or less than about 0.3 mm, or lessthan about 0.4 mm, or less than about 0.5 mm, or less than about 0.6 mm,or less than about 0.7 mm, or less than about 0.8 mm, or less than about0.9 mm, or less than about 1 mm. In another embodiment, the microplatemay have a thickness between about 0.1 mm and 100 mm, or between about0.2 mm and 20 mm, or between about 0.3 mm and 10 mm, or between about0.4 mm and 0.6 mm.

In some embodiments, the coating layer may have a thickness less thanabout 10 micrometers (“microns”), or less than about 5 microns, or lessthan about 1 micron, or less than about 0.5 microns, or less than about0.1 microns.

Another aspect of the invention provides disposable sample holders foruse with polymerase chain reaction (PCR). The disposable sample holdersin some cases are formed of a recyclable material, such as a polymericmaterial, a metallic material (e.g., aluminum), or a composite material.

In some embodiments, a disposable sample holder comprises an aluminumsubstrate coated with a first polymeric material and a plurality ofwells heat-sealed to the first polymeric material. The plurality ofwells can be formed of a second polymeric material compatible with thefirst polymeric material.

In some cases, a disposable sample holder comprises analuminum-containing substrate for providing heat to a plurality of wellsof the disposable sample holder. The disposable sample holder can have aweight less than or equal to about 100 g, or 90 g, or 80 g, or 70 g, or60 g, or 50 g, or 40 g, or 30 g, or 20 g, or 15 g, or 10 g, or 5 g, or 4g, or 3 g, or 2 g, or 1 g, or lower. In some embodiments, the disposablesample holder is a single-use sample holder.

Another aspect of the invention provides a low-cost sample holder foruse with polymerase chain reaction (PCR). The low-cost sample holder cancomprise a substrate formed of a metallic material having a densitybetween about 2.0 g/cm³ and 4.0 g/cm³, or 2.7 g/cm³ and 3.0 g/cm³. Thesubstrate can be configured to provide heat to one or more wells of thelow-cost sample holder at a heating rate between about 0.01° C./secondand 100° C./second, or between about 0.1° C./second and 50° C./second,or between about 1° C./second and 35° C./second, or between about 3°C./second and 25° C./second, or between about 5° C./second and 15°C./second. In some embodiments, the substrate includes aluminum. In somesituations, the low-cost sample holder further includes a barrier layerformed of a first polymeric material over the substrate. The one or morewells of the low-cost sample holder may be formed of a second polymericmaterial joined to the first polymeric material.

FIG. 1 is a schematic cross-sectional side view of a microplate 100, inaccordance with an embodiment of the invention. The microplate 100includes a plurality of wells 101 (or well-like structures) in amoulding 102 comprising one or more tubes formed of a polymericmaterial, such as polypropylene. The tubes are attached to a surface ofa metal plate 103. In some embodiments, the tubes are attached to thesurface of the metal plate 103 with the aid of a coating layer (orbarrier layer) 104 formed of a polymeric material that can be compatiblewith the material of the tubes of the moulding 102. The metal plate maybe formed of an electrically resistive material. In some embodiments,the metal plate may be formed of aluminum or an aluminum alloy. Themicroplate of FIG. 1 has an assay 105 disposed in each of the wells.

In some cases, the moulding 102 can be formed from a single-piecepolymeric material. The moulding 102, in some cases, is formed byinjection moulding. In some situations, the moulding 102 can be formedof a plurality of pieces attached to one another (such as by welding orwith the aid of an adhesive).

With continued reference to FIG. 1, the wells 101 are at least partlydefined by sidewalls of the moulding 102 at least partially formed of apolymeric material. The moulding 102 may have a bottom surface of themoulding resting against the metal plate 103. This can provide forefficient thermal control in each of the wells.

In some embodiments, the moulding 102 can be secured to the metal plate103 with the aid of a bonding material, such as an adhesive. In otherembodiments, the moulding 102 is secured to the metal plate 103 with theaid of a clamp or fastener (not shown).

Microplate Heating

Another aspect of the invention provides a microplate (or consumable)having wells for polymerase chain reaction (PCR) heating. In someembodiments, the consumable can be heated by passing an electricalcurrent through the microplate. The microplate can be heated for apredetermined time period. Sample processing, including heating, can beregulated by a computer system having one or more processors forexecuting machine-readable instructions stored in a memory location ofthe computer system.

Heat can be generated by passing a current through the microplate ofFIG. 1. Heating in some cases is resistive heating. The rate of heatingor cooling can be adjusted by varying the current passing through atleast a portion of the microplate, or varying the electrical potentialapplied across the microplate.

In some embodiments, a disposable microplate (also “consumable” herein)may include a coated metal plate with a polymer moulding attached to themetal plate. The metal plate may be coated with a polymeric materialthat is compatible with the moulding. The polymer moulding may be formedof a polymeric material. The consumable may, in itself, be a heatingelement. The consumable may be directly heated by passing electricalcurrent through the metal plate. The consumable may include liquidsamples or assays that are in close contact with the plate, separatedfrom the plate by a layer of polymer, such that heat transfer to andfrom the samples is fast and controllable. In some embodiments, thelayer of polymer may have a thickness of about 10 microns or otherthickness provided herein (see above).

In some embodiments, the consumable may be heated by passing electricalcurrent through the consumable along a number of different possibleelectric flow paths. In another embodiment, the contact fingers at theends of the plate are connected to a system of bus bars. These bus barsare the single-turn secondary windings of four transformers. Theconsumable is configured to rest on (or come into electrical contactwith) the bus bars. In some embodiments, the consumable is removablefrom the bus bars. In another embodiment, a fixed plate of similargeometry to the described consumable is permanently attached to the busbars.

In some embodiments, the low current primary drive to each transformeris proportionally controlled using phase-angle triggering of triacdevices. Also, by using twin primary windings, the relative phase of thedrive to each transformer can be controlled.

In some embodiments, current passing through the plate are high andvoltage applied to the plate are low. In some embodiments, currentpassing through the plate is up to about 50 A, or 100 A, or 150 A, or200 A, or 300 A, or 400 A, or 500 A, or 600 A, or 700 A, or 800 A, or900 A, or 1000 A per transformer. In another embodiment, voltage appliedto the plate is between about 0.01 volts (“V”) and 20 V, or betweenabout 0.1 V and 10 V, or between about 0.1 V and 1 V, or between about0.25 V and 0.5 V.

In some embodiments, heating is by resistive heating. In some cases,resistive heating is with the aid of direct current (DC). In othercases, resistive heating is with the aid of alternating current (AC).

In some embodiments, in order to operate at low voltage and low plateresistance, contact between the removable plate and the fixed bus barsis critical. In another embodiment, the plate is clamped to gold-platedcontacts on the bus bars using 6 miniature hydraulic rams driven by amaster cylinder actuated by an electric ball screw. The rams may eachexert a force of about 2,000 Newtons (N), which produces sufficientdeformation of the aluminum to disrupt the oxide film typically found onthe surface of that metal, and make very low resistance contacts betweenthe plate and the bus bars.

A microplate can include N rows by M columns of wells, wherein ‘N’ and‘M’ are integers greater than zero. In some cases, N is at least 1, or2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 20, or more, andM is at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10,or 20, or more. The rows can be orthogonal to the columns, or may beangularly disposed in relation to the columns at an angle greater than0° and less than 90° in relation to the columns. For instance, the rowscan be angularly disposed at an angle of about 45° in relation to thecolumns.

For example, a microplate can include 3 rows by 3 columns (3×3) ofwells, or 9 total wells. As another example, a microplate can include3×3, 4×6, 6×4, 9×6 or 6×9 wells. FIG. 10 shows a microplate 1000 having6 rows by 9 columns of wells 1001, or 54 total wells. The wells areformed of a polymeric material and are disposed adjacent to a substrate1002 formed of a metallic material (e.g., aluminum). The substrate 1002comprises a plurality of fingers (or finger-like projections) 1003. Eachfinger 1003 has a top surface (facing the wells 1001) and a bottomsurface. Atop surface of each of the fingers (or micro-plates) 1003 hasa wave pattern that defines a crinkle 1004 on the top surface. A bottomsurface (not shown) of each of the fingers 1003 can have a wave patterndefining a crinkle. At least a portion of the top and bottom surfaces ofthe fingers are configured to come in contact with bus bars forfacilitating the flow of electrical current through the microplate 1000during PCR. In some cases, the crinkles 1004 can be precluded. In othercases, the surfaces of the fingers 1003 are roughened.

In some embodiments, a crinkle has a corrugation between about 0.1micrometers (“microns”) and 1 centimeter, or 1 micron and 10 millimeters(“mm”). In other embodiments, a crinkle has a corrugation of at leastabout 0.1 microns, or 1 micron, or 10 microns, or 100 microns, or 1 mm,or 10 mm, or 100 mm.

In some embodiments, a microplate includes a plurality of wells adjacentto a substrate. The substrate is formed of a metallic material, such asaluminum, and the plurality of wells are at least partly defined by apolymer matrix. In some cases, the polymer matrix defines eachindividual well. In other cases, the polymer matrix defines the one ormore sidewalls of a well, but a bottom portion of a well is defined bythe substrate. In some cases, the bottom portion of a well comprises alayer of a polymeric material adjacent to the substrate.

The microplate includes finger-like projections (see FIG. 10) forenabling the microplate to come in electrical communication with busbars of a system for facilitating the flow of electrical current throughthe microplate. In some cases, a resistance between the microplate andthe plurality of bus bars is minimized, and in some cases renderedohmic, with the aid of wrinkles (or ridges) on surfaces of thefinger-like projections configured to come in contact with the bus bars.The finger-like projections of the microplate can be tightly clamped tothe bus bars.

In some cases, a microplate comprises fingers formed to have a wavepattern on their surfaces, thereby forming a crinkle. The crinkle canaid in removing any oxide layer formed on one or more surfaces of thefingers, which aids in improving the electrical contact between thefingers and the bus bars.

In some cases, a system for facilitating PCR can include a microplate,as described herein, and a temperature sensor for measuring thetemperature in one or more zones of the microplate. The temperaturesensor can be one or more thermocouples in electrical contact with theone or more zones. A thermocouple can be in electrical contact with athermal zone. Alternatively, the temperature sensor can be an infraredsensor for measuring the temperature of one or more zones of themicroplate. The infrared (“IR”) sensor can be a non-contact IR sensorand configured to measure the temperature of a metallic substrate of themicroplate.

The system can include at least 1, or 2, or 3, or 4, or 5, or 6, or 7,or 8, or 9, or 10, or 15, or 20, or 30, or 40, or 50, or 100, or moresensors for measuring the temperature of a microplate. The number ofsensors used for temperature measurements can be equal to the number ofthermal zones in the microplate. For example, the system can includenine sensors for measuring the temperature in each of nine thermal zonesof a microplate.

A temperature sensor can provide continuous measurement of thetemperature in a thermal zone of a microplate. In some cases this canprovide for calibration to deliver a more accurate reading.Alternatively, a temperature sensor can provide intermittent temperaturemeasurements, such as a temperature measurement at least every 0.01seconds, 0.1 seconds, 1 second, 10 seconds, 30 seconds, 1 minute, 10minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 12 hours, 1 day, 2 days, or more. The sensors can providefeedback to determine how much heat is required for a particular zone ofthe plate.

In some embodiments, the temperature variation across a microplate isless than about 10° C., or 5° C., or 1° C., or 0.9° C., or 0.8° C., or0.7° C., or 0.6° C., or 0.5° C., or 0.4° C., or 0.3° C., or 0.2° C., or0.1° C., or lower. This enables the definition of temperature (orthermal) zones for accurate thermal control in each zone.

Microplates provided herein are configured for heating to enable PCR.Some embodiments provided microplates in electrical communication with asource of electrons to enable heating, which may be provided with theaid of an electrical current (“current”) application member. Together, amicroplate, a current application device and any other apparatuses(e.g., bus bars) for bringing the microplate in electrical contact withthe current application device define an electrical flow path, or anelectrical circuit (“circuit”). The current application device can beconfigured for either DC or AC modes of operation.

With reference to FIGS. 2-5, a consumable (center) with 24 wells isprovided, in accordance with an embodiment of the invention. Powersupply units (PSU) are also illustrated. The PSUs may be AC or DC powersupply units. FIGS. 2-5 illustrate various transformer drive patternsfor providing heat to the consumable. In some embodiments, a system isprovided using a 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or20, or 21, or 22, or 23, or 24, or more transformer drive patterns. Insome embodiments, a system is provided using 12 transformer driverpatterns. The arrows associated with the PSUs in FIGS. 2-5 indicate therelative phasing of the active PSUs in the corresponding mode. The PSUor PSUs without an associated arrow are off in that mode. A particularheating pattern is a function of the phasing of each of the PSUs.

With reference to FIG. 2, in a first configuration of relative phasingof PSU1, PSU2, PSU3 and PSU4, heat is provided to a top portion of theconsumable. With reference to FIG. 3, in a second configuration ofrelative phasing of PSU1, PSU2, PSU3 and PSU4, heat is provided to sideportions of the consumable. With reference to FIG. 4, in a thirdconfiguration of relative phasing of PSU1, PSU2, PSU3 and PSU4, heat isprovided to a left side (when looking from the top) of the consumable.With reference to FIG. 5, in a fourth configuration of relative phasingof PSU1, PSU2, PSU3 and PSU4, heat is provided to all or substantiallyall of the consumable.

In embodiments, the heating pattern of a consumable may be the productof a balance between heating rates and cooling rates of the consumable.That is, if the center of the consumable is cooled more rapidly that itis heated, a cooling effect will ensue. If the sides of the consumableare heated more rapidly than the center, the center will remain coolerrelative to the sides of the consumable. In embodiments, heating ratesand cooling may be dependent on various factors, such as, e.g., themodes of heat transfer (i.e., conductive, convective, or radiative) andthe interplay between the modes; heat transfer coefficients; thermalmass; initial temperature; and PSU power.

With reference to FIGS. 2-5, the flow of current may produce apredetermined heating pattern. The use of different current pathsthrough the metal plate may enable use of the consumable as plateheating zones for zonal control, enabling active control of thermaluniformity.

In some embodiments, the plate is cooled from below by means of highpressure air jets, such as 1 or more, or 2 or more, or 3 or more, or 4or more, or 5 or more, or 10 or more high pressure air jets. The jetsmay be switched on and off individually, and air pressure may becontrolled to give proportionality in cooling. This may effectively givezonal control over the applied cooling power. The heating system mayalso be used, even when cooling, to actively maintain overall thermaluniformity. In some embodiments, compressed air may be supplied from abuilding air supply, or a small local compressor, or by using 4miniature air pumps with pulse-width modulation (PWM) control. In allcases the pressure employed is controlled between 0 psi and 50 psi andthe air is directed onto the bottom of the plate by nozzles, such as 4small, 0.7 mm diameter nozzles, which produce high velocity jets topenetrate the boundary layer of the flat plate.

Crinkling of the ends of the plate; the plate when located in themachine not only provides the container for the test samples it also isa resistive heating element. The heating is induced upon the flow of anelectrical current through the aluminum base of the plate. Theconnections between the plate and the rest of the circuit need to be lowresistance when compared to the resistance of the plate so that theinduced heating will not occur in the rest of the circuit. To achieve alow resistance the aluminum fingers (or finger-like projections) of theplate are tightly clamped on to high conductivity bus bars. Suchclamping can provide ohmic contact between the fingers and the bus bars,which can provide for improved heating. In addition to the forcerequired to tightly clamp the fingers, the fingers have been formed tohave a wave pattern in their surface, a crinkle, such that as they areclamped flat there is a wiping action on the surface of the plate whichbreaks down any oxide or contamination that has coated them providing agood connection.

There are a number of elements to this arrangement, such as theprovision of high force, >100 Newtons on a repeatedly made connection.This is achieved by using an over center toggle type clamp; that clampcan have a built-in spring system which reduces the precision needed toset up the clamp. Other clamping methods may be used, such as hydraulicor screw clamping. Slight doming of the clamping ram provides an annularring of contact, rather than a point or face contact which delivers bothhigh contact force and preferable contact area to help providerepeatable low resistance connections. Putting undulations in thesurface of the plate in the area of the clamp enables the material tomove and wipe across clamping surfaces as it is crushed flat by theclamp ram. This process of wiping can used on various connectors toproduce low resistance contacts. In some cases, the preform may becrushed. The size and depth of the preform may be important indetermining the wiping action. With the aid of crinkles, the resistantbetween the microplate and the bus bars can be minimized, and in somecases minimized to below the resistance of an electrical circuit havingthe microplate and a current application device.

In some embodiments, the temperature of the plate may be measured frombelow the plate using a 3×3 array of thermopile-type non-contactsensors. In another embodiment, temperature measurements can be madewith the aid of at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19,or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or30, or 31, or 32, or 33, or 34, or 35, or 36 or more thermopile-typenon-contact sensors. In another embodiment, temperature measurements canbe made with a number of sensors selected to match the number of wells.

FIG. 6 shows sensors on a mounting block, in accordance with anembodiment of the invention. In some situations, the bottom of the platehas an epoxy primer coating to normalize an infrared emissivity of theplate, which may aid in accurate sensor measurement. In othersituations, the bottom of the plate does not include an epoxy primer.Temperature measurements can be made with the aid of a systemoperatively coupled to thermocouples in thermal contact with one or morewells.

In some embodiments, there is no one-to-one mapping between the sensorsand the heating zones. In another embodiment, a computer usesinformation from the sensors to select the optimum transformer drivepattern from the a predetermined number of programmed options, such as12 programmed options. In another embodiment, the transformer drivepattern is updated about 50 times per second. In another embodiment, thetransformer drive pattern is updated at least about 5, or 10, or 20, or30, or 40, or 50, or 60, or 70, or 80, or 90, or 100 times or more persecond.

Infra-red thermopile measurement of temperature; one embodiment uses anarray of non-contact infra-red sensors to measure the temperature of theplate's aluminum base plate. There are 9 sensors in our current arraywhich are used to measure the temperature in nine zones of the plate.These temperatures are used to control the heating system and producethe heating pattern desired. The infra-red sensors are industry standardparts but they only can measure as standard to an accuracy of about 1degree. It is desirable to obtain times that accuracy of measurement;thus one embodiment individually calibrates each sensor across a rangethen uses this information to calculate a more accurate reading. This“calibration” of a sensor requires a number of points to be measured andthese are used to populate an algorithm which extrapolates between themto give a value that is more accurate. This embodiment is advantageousbased at least in part on the use of the “calibration” and the algorithmin combination to deliver a more accurate reading.

Heating control algorithm; the heating system consists of a multi-zoneresistive heating element which can be heated in a number of differentways to provide heat into multiple zones. The temperature of the zonesis measured by an array of non-contact infra-red sensors which providecontinuous measurement. Control of the system is complex because youcan't heat just one zone without heating others both directly by flowingcurrent through the zone and indirectly through heat transfer fromneighboring zones. An algorithm has been developed that provides thiscomplex control using feedback from the thermal sensors to determine howmuch heat is required and where. This algorithm not only gets the plateto the desired temperature quickly it is used to keep the temperaturevariation across the plate to a minimum so that all the test sampleseffectively see the same experimental conditions, important when you aretrying to compare results across test plates and from plate to plate.The novelty here is in the actual nature of the algorithm as well as itsuse.

In some embodiments, a system is provided for controlling heating andcooling of a plate and consumable in thermal communication with theplate. In another embodiment, a system having software is provided forcontrolling heating and cooling of a plate and consumable in thermalcommunication with the plate. In another embodiment, a system isprovided for maintaining thermal uniformity across an active region ofthe plate, whilst following a programmed temperature profile.

In some embodiments, when a user has filled the tubes on the plate withreagents, the tops of the tubes are sealed using a cover, such as atransparent sealing film. This may allow the measurement of fluorescenceto be made from above the plate to follow the progress of PCR. Acharge-coupled device (CCD) camera may be used to record fluorescentoutput. The CCD camera may have a filter wheel. Radiation for excitationmay be provided by one or more excitation sources, such as lightemitting diodes (LED's) with filters.

As an alternative, a microplate may be heated or cooled with the aid ofa heating device employing Peltier heating. In some cases, themicroplate of FIG. 1 may be used with the aid of a Peltier heatingelement in the vicinity of an underside of the microplate. In such acase, the metal plate may permit heat transfer to each of the wells (orchambers) of the microplate. In some cases, a microplate may be inthermal communication with a Peltier heating element, which may transferheat from one side of the heating element to the other side of theheating element against a temperature gradient upon the consumption ofelectrical energy.

FIG. 7 shows a Peltier heating element 700 having a plurality ofsemiconductor-containing elements (or “pellets”) that are chemicallydoped n-type (“N”) 705 or p-type (“P”) 710.

FIG. 8 shows a microplate 800 having a Peltier heating device 801 belowthe microplate 800. The Peltier heating device 801 may include p-type805 and n-type 810 semiconducting (or “semiconductor”) materials, andelectrically conducting material 815 connecting pairs of n-type andp-type semiconductors. The Peltier heating device 801 may include alayer of a thermally insulating material over the n-type and p-typesemiconductors. The layer of thermally insulating material may be aceramic material. The Peltier heating device 801 may provide heating orcooling to the microplate 800, including wells (or chambers) of themicroplate 800. In some cases, the microplate 800 may be heated with theaid of the Peltier heating device 801 in addition to passing a currentthrough the microplate 800, as described above.

As another alternative, the microplate of FIG. 1 may be contacted on anunderside of the microplate (e.g., adjacent to the metal plate of themicroplate) with a resistive, radiative or convective heating device forproviding heating (or cooling) to one or more wells of the microplate.In some cases, the microplate of FIG. 1 may be contacted on theunderside with a clamp heating device. The clamp heating device may beused in conjunction with heating supplied with the aid of currentdirected through the microplate, as described above.

In some cases, heating devices provided herein may be used for bothheating and cooling. For instance, the Peltier heating devices of FIGS.7 and 8 may be used for removing heat from one or more wells of amicroplate by, for example, adjusting the direction of the flow ofcurrent through the semiconductor-containing elements of the Peltierheating devices. As another example, cooling may be provided bydecreasing a heating rate of a heating device, thereby enabling coolingto a pseudo-steady state temperature with the aid of convective,conductive or radiative heat transfer.

Methods for Forming Microplates

Another aspect of the invention provides methods for formingmicroplates. Microplates provided herein can include substrates havingone or more metals. In some embodiments, such substrates can includealuminum, aluminum oxide, an aluminum-containing alloy or compositematerial. In some cases, such substrates include aluminum. Current maybe provided to substrates through electrodes in electrical contact withthe substrates. In some cases, low or substantially low resistanceelectrical contacts may be provided to aluminum substrates for providingcurrent to and through the aluminum substrates.

In some cases, aluminum substrates may be in electrical communicationwith electrical contacts (or electrodes) at a junction resistance lessthan or equal to about 5 m-ohms, or 10 m-ohms, or 15 m-ohms, or 20m-ohms, or 25 m-ohms, or 30 m-ohms, or 35 m-ohms, or 40 m-ohms, wherein1 m-ohm is equal to 1×10⁻⁶ ohms. Such electrical contacts may have a lowconcentration of an aluminum-containing oxide, such as aluminum oxide,AlO_(x), wherein ‘x’ is a number greater than zero.

In some cases, upon manufacturing a microplate having an aluminumsubstrate, corrugations may be pressed of formed in areas of thealuminum substrate for providing electrical contacts to the substrates.For instance, if six electrical contact areas are desired, each of thesix electrical contact areas may corrugated prior to forming theelectrical contact areas. Such corrugation may break any aluminum oxidethat may be formed on a surface of the aluminum substrate, therebyproviding for low or substantially low resistance electrical contacts tothe aluminum substrate.

In some embodiments, one or more components of microplates are formedwith the aid of a die or a plurality of dies. In some cases, microplatesare formed by mechanical cold forming processing, such as forging (e.g.,swaging). For instance, the metal plate of the microplate of FIG. 1 canbe formed using mechanical cold forming processing. In cases in whichthe wells of a microplate are formed of a polymeric material, the wellscan be formed using extrusion or injection molding.

PCR Systems

Another aspect of the invention provides a system for sample processing,including heating for PCR. The system can include a controller with acentral processing, memory (random-access memory and/or read-onlymemory), a data storage unit (e.g., hard drive), a communications port(COM PORTS), and an input/output (I/O) module, such as an I/O interface.The processor may be a central processing unit (CPU) or a plurality ofCPU's for parallel processing. The memory and/or data storage unit canhave machine-readable code for implementing the methods provided herein,such as heating methods for PCR.

FIG. 9 shows a system 900 for regulating PCR using microplates providedherein, in accordance with an embodiment of the invention. The system900 includes a processor 901, memory 902, input/output module 903,communications interface 904 and data storage unit 905. The system 900can be operatively coupled to a display 906 for presenting a userinterface 907 to a user operating the system 900. The user interface 907in some cases is a graphical user interface (GUI) having one or moretextual, graphical, audio and video elements. The display 906 can be atouch screen, such as a capacitive touch or resistive touch screen. Insome embodiments, the display 906 is disposed adjacent to the system900. In other embodiments, the display 906 is disposed remotely from thesystem 900.

The system 900 is operatively coupled to a PCR system 908 for performingPCR using microplates provided herein. The PCR system 908 can includesensors (e.g., thermocouples) for enabling the system 900 to maketemperature measurements during PCR with the aid of the PCR system 908.

The memory 902 can be random-access memory (RAM) or read-only memory(ROM), to name a few examples, or a hard drive. The memory can includemachine-readable code for implementing a method for performing PCR usingthe PCR system 908. In some embodiments, the memory 902 includesmachine-readable code for executing one or more temperature profiles,which can include temperature zone profiles as a function of time.

In an example, a user inputs a PCR microplate having a sample into thePCR system 908. The PCR microplate can be as described herein. With theaid of the user interface 907 of the display, the user requests that thesystem 900 initiate sample processing and perform PCR on the sample. Thesystem 900 executes code stored on the memory 902 to provide aprogrammed temperature profile (e.g., ramp rate) to the sample toconduct PCR.

The system 900 can be in wired or wireless communication with a remotesystem for housing data or providing instructions for PCR (see below).Communication to and from the system can be facilitated by a networkinterface that brings the system and in communication with the remotesystem through an intranet or the Internet (e.g., the World Wide Web).

Aspects of the systems and methods provided herein may be embodied inprogramming. Various aspects of the technology may be thought of as“products” or “articles of manufacture” typically in the form ofexecutable code and/or associated data that is carried on or embodied ina type of machine-readable medium. “Storage” type media may include anyor all of the tangible memory of the computers, processors or the like,or associated modules thereof, such as various semiconductor memories,tape drives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server or an intensity transformsystem. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine-readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

Another aspect of the invention provides a method for conducting PCR inwhich one or more of data from the reaction (e.g., fluorescenceinformation, measured temperature), instructions for conducting PCR(e.g., ramp rate, predetermined temperature profile) and instructionsfor processing the data are located on a microplate, remotely or on aremovable device. This can enable for plug-and-play PCR in which PCR canbe performed across various platforms without the need for additionalsetup.

In some cases, a removable device can be configured to interface withsystems for conducting PCR, such as the system 900 of FIG. 9. In anexample, the removable device is a universal serial bus (USB) drive(e.g., USB stick), or a removable memory disk (e.g., flash drive). Inanother example, the removable disk is a compact flash disk, or deviceconfigured to communicate with a serial advanced technology attachmentinterface (e.g., mini SATA, or M-SATA) or a personal computer memorycard international association (PCMCIA, also PC card) interface.

In some situations, both control and analysis instructions are providedon the removable device to allow a user to develop an experiment andanalyze the results independently from a thermal cycler used forconducting the PCR reaction. Machine-readable instructions forimplementing PCR can be located on the removable device. In someembodiments, the removable disk includes instructions and/or commands(e.g., as embodied in machine-readable code) that enable anidentification of the type of hardware (or system, such as the system900) interfacing with the removable device. The removable device caninclude processing instructions for performing PCR on the hardware. Theprocessing instructions can be predetermined based on the type of systemcoupled to the removable device and/or the type of sample. The removabledevice can help identify the type of hardware it is plugged into andprovide predetermined commands/interfaces to conduct PCR on thathardware directly without having to be installed on the hardware.

Some embodiments provide a removable device and software located on aremovable device that is configured to operate on various platforms.Test system software houses both control and analysis programs so thatthe user can develop the user's experiment and understand the results.Whilst operating on the machine itself it is also desirable that it willoperate remotely to enable experimental design and results analysis tooccur away from the test system. This software can reside on a removabledevice, such as a USB stick, other removable memory disks, such as, forexample, a compact flash, M-SATA, or PCMCIA device.

Such systems and devices provide various advantages. For example, havingcommands and/or instructions on a removable device can preclude the needfor any additional installation. PCR can be conducted in such caseswithout the need for administrator privileges, and it be can performedon a machine without having to be installed on that machine. Thisprovides a uniform platform for sample processing, as no hardware and/orsoftware upgrades or installation may be required to setup a system(e.g., system 900) for PCR on a particular sample. The removable mediacan store both the data files and the program so as to enablecompatibility.

PCR systems provided herein are configured for installation andoperation on various software platforms, such as Windows-based (e.g.,Windows 7) and Linux-based (e.g., Mac OS X) operating systems. Systemsprovided herein can be implemented on portable electronic devices, suchas laptop computers, Smartphone (e.g., Apple iPhone®) and tablets (e.g.,Apple iPad®). In some cases, such systems can communicate withperipheral devices for PCR, such as a heating system (e.g., currentapplication device in communication with a microplate to define acircuit). This can provide for an interface for ready recognition acrossvarious platforms.

PCR systems provided herein can be platform independent. In somesituations, as long as the system can accept the removable memorydevice, then it would be able to run the software and conduct PCR. Insome cases, all the information is stored on the removable device suchthat nothing is held on the platform that is running the software, whichmay reduce, if not eliminate, data security issues. The data and theapplication are transferred from the removable device, and the systemprovides the computing power and associated ancillary functions, such asa user interface and printing.

Alternatively, PCR commands and/or instructions are stored a remoteserver (i.e., the “cloud”) and accessed by the system (e.g., the system900) through a network interface, such as a wired or wireless interface.A user can run PCR by providing a microplate, as described herein havinga sample, and using the system to retrieve the requisite instructionsfor conducting PCR. Data gathered through the course of PCR can bestored on the system and subsequently uploaded to the remote serverhaving a data storage unit.

Alternatively, PCR commands and/or instructions are stored on a memorydevice that is integrated in a microplate. The microplate is configuredto interface with a system for conducting PCR, such as the system 900 ofFIG. 9. The system can include a reader for recognizing the memorydevice and subsequently preparing the system for sample processing. Insome embodiments, the memory device is an electrically erasableprogrammable read-only memory (EEPROM).

In some embodiments, a microplate (or sample holder) includes anidentification member for enabling a system (e.g., the system 900 ofFIG. 9) to identify the microplate. The identification member can be asolid state device, such as a processor (e.g., microprocessor) ormemory, or a radio-frequency identification (RFID) tag or device. Theidentification member can be for identification and in some cases datastorage. This can advantageously aid in reducing handling errors. In anexample, a microplate includes an RFID device that stores bothinstructions (e.g., PCR instructions, data processing instructions) andidentifying information. The identifying information can be a serialnumber. The identifying information can enable a system to identify themicroplate. In some situations, the RFID device only includesidentifying information, and a system, such as the system 900 of FIG. 9,is configured to detect the RFID and retrieve the identifyinginformation, and to retrieve instructions for conducting PCR with theaid of the microplate. In some cases, the instructions retrieved by thesystem are specific to the microplate operatively coupled to the system.

User Interface

Another aspect of the invention provides a user interface for enabling auser to setup a system for PCR, monitor PCR, and view PCR results. Theuser interface in some cases is a graphical user interface (GUI) havingone or more textual, graphical, audio and video elements, such as menuelements for enabling a user to setup a PCR task, monitor the course ofPCR, review results and conduct data analysis. The GUI can facilitatethe implementation of PCR experiments. FIGS. 11-17 illustratescreenshots of a GUI that allows ready analysis and visualization of PCRresults, as described herein. The GUI of FIGS. 11-17 can be implementedon systems provided herein, such as the system 900 of FIG. 9, andpresented to a user with the aid of a display, such as the display 906of FIG. 9.

FIG. 11 shows a GUI having various menu options, including “START ATEST” and “ANALYSIS.” A user can select the START A TEST option toinitiate PCR of a sample in a microplate coupled to a system having theGUI. The user can select ANALYSIS to analyze PCR data. The menu optionscan be selected with the aid of a pointing device, such as a mouse orthe user's finger in cases in which the GUI is displayed on a touchscreen.

In FIG. 12, the user has selected START A TEST and the system presentsthe user with sub-menu options, including TEMPLATES and HISTORY. Theuser can select TEMPLATES to access various test templates forperforming PCR. Alternatively, the user can select HISTORY to review,for example, PCR history, such as what tests were conducted at aparticular point in time. Such data can be stored in a data repositoryof the system. The user can select STANDARD TEST to proceed with settingup the system to conduct PCR.

With reference to FIG. 13, under STANDARD TEST, the system presents theuser with various PCR options, such requesting that the user select thetype of PCR (“SELECT PCR TYPE”). The system presents the user withvarious PCR type options, including RICTOR, PKC, Rac, Rho, Akt and RHEB.The user can select a PCR type option. Next, with reference to FIG. 14,the system presents the user with various chemistry types (“CHEMISTRYTYPE”), such as ABI, Fast Advanced Master Mix and Gene Expression MasterMix. Next, with reference to FIG. 15, the system presents the user withtray settings (“TRAY SETTINGS”) and enables the user to select genes ofinterest (“GENES OF INTEREST”), such as mTOR or RAPTOR. Next, withreference to FIG. 16, the system presents the user with thermal profileoptions, including start temperatures and target temperatures. The usercan select from predetermined options, or manually input temperaturesettings. The system then presents the user with a confirmation screen,as shown in FIG. 17. The confirmation screen shows a temperature profileper cycle that the system will use in the PCR, the number of cycles(“REPEAT”), tray settings (“24 WELLS” has been selected in FIG. 17),among other settings. The temperature profile is a custom temperatureprofile, though predetermined settings may be used, if desired. Thesystem provides the user the option to change the settings, or toproceed with conducting PCR (“RUN TEST”).

Example 1 Coated Metal Plate

Nominally 0.4 mm thick metal plates were produced from bulk processedmaterial on a large scale where a metal ingot (e.g., 5 ton metal ingot)enters the process and is rolled and coated in a continuous operation.The material was an aluminum alloy rolled to a half-hard condition andthen coated on one side (e.g., a top side) to a nominal thickness ofabout 10 microns with a polypropylene compatible material. This materialallows polypropylene to be heat-sealed (or welded) to the metal plate,and does not inhibit the PCR. The other side (bottom) of the sheet wascoated with an epoxy primer to a nominal thickness of 5 microns. This ispresent to normalize the infrared emissivity of the bottom side of thesheet. The material was slit into 160 mm wide strips and supplied incoiled form to an automatic stamping line where the individual platesare produced. The epoxy coating was then selectively removed from thecontact fingers at the ends of the plates to allow electrical contact tobe made.

Example 2 Polypropylene Moulding

To contain the liquid samples placed on the plate, a polypropylenemoulding consisting of an array of vertical tube structures was weldedto the metal plate of Example 1. The polypropylene moulding was formedof a plurality of tubes to define sample areas (or wells). The size andpattern of the tubes may be a matter of user choice; any pattern thatfits within the actively temperature-controlled area in the middle ofthe plate may be used. Two familiar-looking options were selected: a 6×4tube array on a 9 mm pitch, and an 8×12 array on a 4.5 mm pitch. Thewhole assembly weighed 10.5 g and was readily recyclable. Appropriatelyfor a single-use item, the manufacturing cost of the consumable was low.

While certain microplates have been describes as being consumable orerecyclable, it will be appreciated that in some cases such microplatesneed not be consumable or recyclable. In some embodiments, suchmicroplates may be reusable, non-consumable, or non-recyclable.

Systems and methods provided herein may be combined with or modified byother systems and methods. For example, systems and methods providedherein may be combined with or modified by systems and methods describedin U.S. Pat. No. 6,635,492 to Gunter (“Heating specimen carriers”) andU.S. Pat. No. 6,949,725 to Gunter (“Zone heating of specimen carriers”),and PCT Publication Nos. WO/2001/072424 to Gunter (“Heating specimencarriers”), WO/1997/026993 to Gunter (“Heating”), WO/2005/058501 toGunter (“Heating samples in specimen carriers”) and WO/2003/022439 toGunter (“Zone heating of specimen carriers”), which patents and patentpublications are entirely incorporated herein by reference.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents.

What is claimed is:
 1. A method for processing a biological sample,comprising: (a) providing a microplate comprising (i) a substrate havinga metallic material, (ii) a barrier layer coated on said substrate, saidbarrier layer formed of a first polymeric material, (iii) a mouldingcomprising one or more wells sealed to said barrier layer, said one ormore wells formed of a second polymeric material that is separate fromsaid first polymeric material, and (iv) electrodes as projections fromsaid substrate in electrical communication with said substrate; (b)depositing said biological sample in a solution in said one or morewells; and (c) directing electrical current from said electrodes throughsaid substrate to subject said solution to one or more heating cycles,which one or more heating cycles comprise heating said solution at aheating rate of at least about 5° C./second upon flow of said electricalcurrent through said substrate.
 2. The method of claim 1, wherein saidelectrodes have corrugated surfaces.
 3. The method of claim 1, furthercomprising bringing said electrodes in electrical communication with apower bus, which power bus provides said electrical current to saidelectrodes.
 4. The method of claim 1, wherein said electrodes comprise afirst electrode and a second electrode as projections on different sidesof said substrate.
 5. The method of claim 1, wherein said solution issubjected to said one or more heating cycles at a rate of at least abouttwo cycles per minute.
 6. The method of claim 1, further comprisingmeasuring a temperature of said solution using one or more temperaturesensors in thermal communication with said solution.
 7. The method ofclaim 6, wherein said one or more temperature sensors include infraredsensors.
 8. The method of claim 6, wherein said temperature is measuredat least every 0.01 seconds.
 9. The method of claim 1, wherein saidmicroplate further comprises a plurality of thermal zones.
 10. Themethod of claim 9, wherein said plurality of thermal zones includes ninezones.
 11. The method of claim 9, wherein said microplate furthercomprises a multi-zone resistive heating element that provides heat tosaid plurality of thermal zones.
 12. The method of claim 9, furthercomprising controlling said heating rate by determining an amount ofheating in each of said plurality of thermal zones based on one or moretemperature measurements from one or more temperature sensors.
 13. Themethod of claim 1, wherein said electrical current is directed through aplurality of electrical paths for zonal heating control of saidmicroplate.
 14. A method for processing a biological sample, comprising:(a) providing a microplate comprising (i) a substrate having a metallicmaterial, (ii) a barrier layer coated on said substrate, said barrierlayer formed of a first polymeric material, (iii) a moulding comprisingone or more wells sealed to said barrier layer, said one or more wellsformed of a second polymeric material that is separate from said firstpolymeric material, and (iv) electrodes as projections from saidsubstrate in electrical communication with said substrate; (b)depositing said biological sample in a solution in said one or morewells; and (c) directing electrical current from said electrodes throughsaid substrate to subject said solution to one or more heating cycles ata rate of at least about 1 heating cycle per 30 seconds.
 15. The methodof claim 14, wherein said electrodes have corrugated surfaces.
 16. Themethod of claim 14, further comprising bringing said electrodes inelectrical communication with a power bus, which power bus provides saidelectrical current to said electrodes.
 17. The method of claim 14,wherein said electrodes comprise a first electrode and a secondelectrode as projections on different sides of said substrate.
 18. Themethod of claim 14, further comprising measuring a temperature of saidsolution using one or more temperature sensors in thermal communicationwith said solution.
 19. The method of claim 14, wherein said microplatefurther comprises a plurality of thermal zones.
 20. The method of claim19, wherein said microplate further comprises a multi-zone resistiveheating element that provides heat to said plurality of thermal zones.21. The method of claim 19, further comprising controlling said heatingrate by determining an amount of heating in each of said plurality ofthermal zones based on one or more temperature measurements from one ormore temperature sensors.
 22. The method of claim 14, wherein saidelectrical current is directed through a plurality of electrical pathsfor zonal heating control of said microplate.